JNK (1-3) are members of the stress-activated protein kinase family that regulates the cellular stress response via their control of cell death and survival. Our recent studies disclose an unexpected mechanism for the regulation and consequently the function of JNK; our preliminary results demonstrate that through its KEN-box, JNK is targeted for degradation in a cell cycle-dependent manner. Such targeting is mediated by Cdh1, which recruits the Anaphase Promoting Complex or Cyclosome (APC/C) ubiquitin ligase to ubiquitinate JNK, resulting in its proteasome-dependent degradation. APC/CCdh1-mediated JNK degradation primarily occurs during the exit from mitosis, and G1 phases of the cell cycle. Our preliminary data also identify Cdc25C as JNK substrate at the G2/M-G1 phases of the cell cycle. JNK degradation is important for the regulation of Cdc25C activity and Wee1 stability. Thus, a non-degradable (yet which can be activated) form of JNK (JNKKEN) induces reduced cyclin-B/Cdk1 activity, affects spindle and chromosomal dynamics and delays exit from mitosis. These findings provide the foundation for our hypothesis that through the regulation of Cdc25C, JNK plays an important role in control of cell cycle progression during which it is tightly regulated by the APC/CCdh1. Our finding that JNK is a cell cycle regulator offers an undisclosed link between JNK and unrestrained cell cycle progression as well as chromosome instability, commonly observed in human cancers, where the balance between JNK functions as a stress kinase and cell cycle control protein might be altered. To test our hypothesis we propose to carry out the following studies: Aim 1 - Assess the molecular determinants responsible for JNK activity during the cell cycle and determine which of JNK family members is most important for cell cycle control.; Aim 2- characterize the effect of JNK on Cdc25C as part of its control of cell cycle transition. Aim 3 -Characterize the cytological changes in mitosis that are controlled by JNK and Cdc25C regulation by JNK; Aim 4 - determine the physiological significance of JNK's role in cell cycle control in a JNK-KEN knock-in mouse model. Aim 5 - using genetic, biochemical and cell biology approaches, determine the role of JNK in cell cycle checkpoint pathways following stress and DNA damage and in human melanoma as a model for constitutively deregulated JNK activity. This proposal brings together experts that closely collaborate to address an important and novel function of the key kinase, JNK. This concerted effort will provide the foundation for understanding the role of JNK in cell cycle progression and the implication of its deregulated control to genotoxic stress response as well as to development of human cancer. PUBLIC HEALTH RELEVANCE: This application will test the hypothesis that JNK is important player in cell cycle control prior and more so, in response to DNA damage. Support for this hypothesis comes from studies in which we discovered that JNK is targeted for degradation at G2M-G1 phases of the cell cycle. JNK mediates its function through phosphorylation of Cdc25C which affects its phosphatase activity. Interfering with JNK degradation or altering its activity delays exit from mitosis and impairs G2 arrest after DNA damage. Using biochemical, cellular and genetic models our proposed studies will delineate the requirements and regulation of this newly identified regulatory lobe along the cell cycle and DNA damage response.